Human pyrimidine receptor

ABSTRACT

This invention provides a receptor having a preference for pyrimidine nucleotides preferably uridine triphosphate over purine nucleotides. A receptor having a preference for pyrimidine nucleotides over purine nucleotides means a receptor for which pyrimidine nucleotides and purine nucleotides are not equally active and equipotent. This means that the receptor according to the invention in presence of these agonists presents a functional response (preferably the accumulation of Inositol triphosphate (IP3), diacylglycerol (DAG), or calcium ions) to lower concentration of pyrimidine nucleotides, preferably uridine triphosphate, than to purine nucleotides or a more important functional response to similar concentration of pyrimidine nucleotide than to purine nucleotide

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/077,173,filed Nov. 12, 1998 now U.S. Pat. No. 6,790,626 which is the U.S.National stage of International Application No. PCT/BE96/00123, filed onNov. 21, 1996, which designated the United States and was published inEnglish, which claims priority to foreign application EP 95870124.5filed on Nov. 21, 1995. The entire teachings of the above application(s)are incorporated herein by reference.

OBJECT OF THE PRESENT INVENTION

The present invention concerns a new receptor having a preference forpyrimidine nucleotides preferably uridine triphosphate over purinenucleotides and the nucleic acid molecule encoding said receptor,vectors comprising said nucleic acid molecule, cells transformed by saidvector, antibodies directed against said receptor, nucleic acid probesdirected against said nucleic acid molecule, pharmaceutical compositionscomprising said produces and non human transgenic animals expressing thereceptor according to the invention or the nucleic acid moleculeaccording to said receptor.

The invention further provides methods for determining ligand binding,detecting expression, screening for drugs, molecular bindingspecifically to said receptor and treatment involving the receptoraccording to the invention.

BACKGROUND

The cloning of several receptors for ATP has been reported since 1993.In keeping with the latest nomenclature proposal, these P2 purinergicreceptors can be subdivided into two classes: G protein-coupledreceptors, or P2Y receptors, and receptors with intrinsic ion channelactivity or P2X receptors (2). Two distinct rat P2X receptors have beencloned, respectively from the vas deferens (3) and phaechromocytoma PC12cells (4): they have a characteristic topology, with two hydrophobicputatively membrane-spanning segments and an ion pore motif reminiscentof potassium channels. In the P2Y family, the sequences of two subtypes,both coupled to phospholipase C, have been published: chick (5), turkey(6), bovine (7), mouse and rat (8) P2Y1 receptors (formerly called P2Y);murine (9, 10), rat (11) and human (12) P2Y2 receptors (previously namedP2U) on the other hand. In addition, a P2Y3 receptor, with a preferencefor ADP over ATP, has been cloned from chick brain, but its sequence isnot yet published (13). Furthermore, the 6H1 orphan receptor, clonedfrom activated chicken T lymphocytes, exhibits a significant degree ofhomology to the P2Y1 and P2Y2 receptors, suggesting that it also belongsto the P2Y family, although its responsiveness to nucleotides has notyet been demonstrated (14).

SUMMARY OF THE INVENTION

This invention provides a receptor having a preference for pyrimidinenucleotides preferably uridine triphosphate over purine nucleotides. Areceptor having a preference for pyrimidine nucleotides over purinenucleotides means a receptor for which pyrimidine nucleotides and purinenucleotides are not equally active and equipotent. This means that thereceptor according to the invention in presence of these agonistspresents a functional response (preferably the accumulation of Inositoltriphosphate (IP3), diacylglycerol (DAG), or calcium ions) to lowerconcentration of pyrimidine nucleotides, preferably uridinetriphosphate, than to purine nucleotides or a more important functionalresponse to similar concentration of pyrimidine nucleotide than topurine nucleotide.

The inositol phosphate (IP3) accumulation after addition of saidagonists is described in the specification thereafter.

Advantageously, the receptor according to the invention has at least atwofold, preferably a tenfold to one hundredfold preference forpyrimidine nucleotides over purine nucleotides.

A preferred embodiment of the receptor according to the invention ischaracterized by a preference for uridine triphosphate over adeninenucleotides.

The receptor according to the invention is a receptor, preferably a Gprotein-coupled receptor, which belongs structurally to the purinergicreceptor family (P2Y family) but functionally is a pyrimidinergicreceptor, preferably a UTP-specific receptor.

According to a preferred embodiment of the present invention, thereceptor is a human receptor.

Said receptor has an amino acid sequence having more than 60% homologywith the amino acid sequence shown in FIG. 1. Preferably, the amino acidsequence of the receptor according to the invention has at least theamino acid sequence shown in FIG. 1 or a portion thereof.

A portion of the amino acid sequence means a peptide or a protein havingthe same binding properties as the receptor according to the invention(i.e. peptide or a protein which is characterized by a preference forpyrimidine nucleotides, preferably UTP, over purine nucleotides).

The present invention is also related to a nucleic acid molecule, suchas a DNA molecule or an RNA molecule, encoding the receptor according tothe invention.

Preferably, said DNA molecule is a cDNA molecule or a genomic DNAmolecule.

Preferably, the nucleic acid molecule according to the invention is atleast the DNA sequence shown in FIG. 1 or portion thereof. “A portion ofa nucleic acid sequence” means a nucleic acid sequence encoding at leasta portion of amino acid sequence as described above.

The present invention is also related to a vector comprising the nucleicacid molecule according to the invention. Preferably, said vector isadapted for expression in a cell and comprises the regulatory elementsnecessary for expressing the amino acid molecule in said celloperatively linked to the nucleic acid sequence according to theinvention as to permit expression thereof.

Preferably, said cell is chosen among the group consisting of bacterialcells, yeast cells, insect cells or mammalian cells. The vectoraccording to the invention is a plasmid or a virus, preferably abaculovirus, an adenovirus or a semliki forest virus.

The plasmid may be the pcDNA3-P2Y4.

The present invention concerns also the cell (preferably a mammaliancell, such as a 1321N1 cell) transformed by the vector according to theinvention. Advantageously, said cell is preferably non neuronal inorigin and is chosen among the group consisting of a COS-7 cell, anLM(tk-) cell, an NIH-3T3 cell or a 1321N1 cell.

The present invention is also related to a nucleic acid probe comprisingthe nucleic acid molecule according to the invention, of at least 15nucleotides capable of specifically hybridizing with a unique sequenceincluded in the sequence of the nucleic acid molecule encoding thereceptor according to the invention. Said nucleic acid probe may be aDNA or an RNA molecule.

The invention concerns also an antisense oligonucleotide having asequence capable of specifically hybridizing to an mRNA moleculeencoding the receptor according to the invention so as to preventtranslation of said mRNA molecule or an antisense oligonucleotide havinga sequence capable of specifically hybridizing to the cDNA moleculeencoding the receptor according to the invention.

Said antisense oligonucleotide may comprise chemical analogs ofnucleotide or substances which inactivate mRNA, or be included in an RNAmolecule endowed with ribozyne activity.

Another aspect of the present invention concerns a ligand other thanpurine and pyrimidine nucleotides (preferably an antibody) capable ofbinding to a receptor according to the invention and an anti-ligand(preferably also an antibody) capable of competitively inhibiting thebinding of said ligand to the receptor according to the invention.

Preferably, said antibody is a monoclonal antibody.

The present invention concerns also the monoclonal antibody directed toan epitope of the receptor according to the invention and present on thesurface of a cell expressing said receptor.

The invention concerns also the pharmaceutical composition comprising aneffective amount of oligonucleotide according to the invention,effective to decrease the activity of said receptor by passing through acell membrane and binding specifically with mRNA encoding the receptoraccording to the invention in the cell so as to prevent its translation.The pharmaceutical composition comprises also a pharmaceuticallyacceptable carrier capable of passing through said cell membrane.

Preferably, in said pharmaceutical composition, the oligonucleotide iscoupled to a substance, such as a ribozyme, which inactivates mRNA.

Preferably, the pharmaceutically acceptable carrier comprises astructure which binds to a receptor on a cell capable of being taken upby cell after binding to the structure. The structure of thepharmaceutically acceptable carrier in said pharmaceutical compositionis capable of binding to a receptor which is specific for a selectedcell type.

Preferably, said pharmaceutical composition comprises an amount of theantibody according to the invention effective to block the binding of aligand to the receptor according to the invention and a pharmaceuticallyacceptable carrier.

The present invention concerns also a transgenic non human mammaloverexpressing (or expressing ectopically) the nucleic acid moleculeencoding the receptor according to the invention.

The present invention also concerns a transgenic non human mammalcomprising a homologous recombination knockout of the native receptoraccording to the invention.

According to a preferred embodiment of the invention, the transgenic nonhuman mammal whose genome comprises antisense nucleic acid complementaryto the nucleic acid according to the invention is so placed as to betranscripted into antisense mRNA which is complementary to the mRNAencoding the receptor according to the invention and which hybridizes tomRNA encoding said receptor, thereby reducing its translation.Preferably, the transgenic non human mammal according to the inventioncomprises a nucleic acid molecule encoding the receptor according to theinvention and comprises additionally an inducible promoter or a tissuespecific regulatory element.

Preferably, the transgenic non human mammal is a mouse.

The invention relates to a method for determining whether a ligand canbe specifically bound to the receptor according to the invention, whichcomprises contacting a cell transfected with a vector expressing thenucleic acid molecule encoding said receptor with the ligand underconditions permitting binding of ligand to such receptor and detectingthe presence of any such ligand bound specifically to said receptor,thereby determining whether the ligand binds specifically to saidreceptor.

The invention relates to a method for determining whether a ligand canspecifically bind to a receptor according to the invention, whichcomprises preparing a cell extract from cells transfected with a vectorexpressing the nucleic acid molecule encoding said receptor, isolating amembrane fraction from the cell extract, contacting the ligand with themembrane fraction under conditions permitting binding of the ligand tosuch receptor and detecting the presence of any ligand bound to saidreceptor, thereby determining whether the compound is capable ofspecifically binding to said receptor. Preferably, said method is usedwhen the ligand is not previously known.

The invention relates to a method for determining whether a ligand is anagonist of the receptor according to the invention, which comprisescontacting a cell transfected with a vector expressing the nucleic acidmolecule encoding said receptor with the ligand under conditionspermitting the activation of a functional receptor response from thecell and detecting by means of a bio-assay, such as a modification in asecond messenger concentration or a modification in the cellularmetabolism (preferably determined by the acidification rate of theculture medium), an increase in the receptor activity, therebydetermining whether the ligand is a receptor agonist.

The invention relates to a method for determining whether a ligand is anagonist of the receptor according to the invention, which comprisespreparing a cell extract from cells transfected with a vector expressingthe nucleic acid molecule encoding said receptor, isolating a membranefraction from the cell extract, contacting the membrane fraction withthe ligand under conditions permitting the activation of a functionalreceptor response and detecting by means of a bio-assay, such as amodification in the production of a second messenger an increase in thereceptor activity, thereby determining whether the ligand is a receptoragonist.

The present invention relates to a method for determining whether aligand is an antagonist of the receptor according to the invention,which comprises contacting a cell transfected with a vector expressingthe nucleic acid molecule encoding said receptor with the ligand in thepresence of a known receptor agonist, under conditions permitting theactivation of a functional receptor response and detecting by means of abio-assay, such as a modification in second messenger concentration or amodification in the cellular metabolism, (preferably determined by theacidification rate of the culture medium) a decrease in the receptoractivity, thereby determining whether the ligand is a receptorantagonist.

The present invention relates to a method for determining whether aligand is an antagonist of the receptor according to the invention,which comprises preparing a cell extract from cells transfected with anexpressing the nucleic acid molecule encoding said receptor, isolating amembrane fraction from the cell extract, contacting the membranefraction with the ligand in the presence of a known receptor agonist,under conditions permitting the activation of a functional receptorresponse and detecting by means of a bio-assay, such as a modificationin the production of a second messenger, a decrease in the receptoractivity, thereby determining whether the ligand is a receptorantagonist.

Preferably, the second messenger assay comprises measurement ofintracellular cAMP, intracellular inositol phosphate (IP3),intracellular diacylglycerol (DAG) concentration or intracellularcalcium mobilization.

Preferably, the cell used in said method is a mammalian cell nonneuronal in origin, such as a COS-7 cell, a CHO cell, a LM(tk-) cell anNIH-3T3 cell or 1321N1.

In said method, the ligand is not previously known.

The invention is also related to the ligand isolated and detected by anyof the preceding methods.

The present invention concerns also the pharmaceutical composition whichcomprises an effective amount of an agonist or an antagonist of thereceptor according to the invention, effective to reduce the activity ofsaid receptor and a pharmaceutically acceptable carrier.

For instance, said agonist or antagonist may be used in a pharmaceuticalcomposition in the treatment of cystic fibrosis, and the methodaccording to the invention may be advantageously used in the detectionof improved drugs which are used in the thereby determining whether theligand is a receptor antagonist.

The present invention relates to a method for determining whether aligand is an antagonist of the receptor according to the invention,which comprises preparing a cell extract from cells transfected with anexpressing the nucleic acid molecule encoding said receptor, isolating amembrane fraction from the cell extract, contacting the membranefraction with the ligand in the presence of a known receptor agonist,under conditions permitting the activation of a functional receptorresponse and detecting by means of a bio-assay, such as a modificationin the production of a second messenger, a decrease in the receptoractivity, thereby determining whether the ligand is a receptorantagonist.

Preferably, the second messenger assay comprises measurement ofintracellular cAMP, intracellular inositol phosphate (IP3),intracellular diacylglycerol (DAG) concentration or intracellularcalcium mobilization.

Preferably, the cell used in said method is a mammalian cell nonneuronal in origin, such as a COS-7 cell, a CHO cell, a LM(tk-) cell anNIH-3T3 cell or 1321N1.

In said method, the ligand is not previously known.

The invention is also related to the ligand isolated and detected by anyof the preceding methods.

The present invention concerns also the pharmaceutical composition whichcomprises an effective amount of an agonist or an antagonist of thereceptor according to the invention, effective to reduce the activity ofsaid receptor and a pharmaceutically acceptable carrier.

For instance, said agonist or antagonist may be used in a pharmaceuticalcomposition in the treatment of cystic fibrosis, and the methodaccording to the invention may be advantageously used in the detectionof improved drugs which are used in the treatment of cystic fibrosis.

Therefore, the previously described methods may be used for thescreening of drugs to identify drugs which specifically bind to thereceptor according to the invention.

The invention is also related to the drugs isolated and detected by anyof these methods.

The present invention concerns also a pharmaceutical compositioncomprising said drugs and a pharmaceutically acceptable carrier.

The invention is also related to a method of detecting expression of areceptor according to the invention by detecting the presence of mRNAcoding for a receptor, which comprises obtaining total RNA or total mRNAfrom the cell and contacting the RNA or mRNA so obtained with thenucleic acid probe according to the invention under hybridizingconditions and detecting the presence of mRNA hybridized to the probe,thereby detecting the expression of the receptor by the cell.

Said hybridization conditions are stringent conditions.

The present invention concerns also the use of the pharmaceuticalcomposition according to the invention for the treatment and/orprevention of cystic fibrosis.

The present invention concerns also a method for diagnosing apredisposition to a disorder associated with the activity of thereceptor according to the invention. Said method comprises:

a) obtaining nucleic acid molecules of subjects suffering from saiddisorder;

b) performing a restriction digest of said nucleic acid molecules with apanel of restriction enzymes;

c) electrophoretically separating the resulting nucleic acid fragmentson a sized gel;

d) contacting the resulting gel with a nucleic acid probe capable ofspecifically hybridizing to said nucleic acid molecule and labeled witha detectable marker;

e) detecting labeled bands which have hybridized to the said nucleicacid molecule labeled with a detectable marker to create a unique bandpattern specific to subjects suffering from said disorder;

f) preparing nucleic acid molecules obtained for diagnosis by step a-e;and

g) comparing the unique band pattern specific to the nucleic acidmolecule of subjects suffering from the disorder from step e and thenucleic acid molecule obtained for diagnosis from step f to determinewhether the patterns are the same or different and to diagnose therebypredisposition to the disorder if the patterns are the same.

A last aspect of the present invention concerns a method of preparingthe receptor according to the invention, which comprises:

a) constructing a vector adapted for expression in a cell whichcomprises the regulatory elements necessary for the expression ofnucleic acid molecules in the cell operatively linked to nucleic acidmolecule encoding said receptor so as to permit expression thereof,wherein the cell is selected from the group consisting of bacterialcells, yeast cells, insect cells and mammalian cells;

b) inserting the vector of step a in a suitable host cell;

c) incubating the cell of step b under conditions allowing theexpression of the receptor according to the invention;

d) recovering the receptor so obtained; and

e) purifying the receptor so recovered, thereby preparing an isolatedreceptor according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the nucleotide and deduced amino acid sequence of ahuman P2Y₄ receptor according to the invention. SEQ ID NO:1 and SEQ IDNO:2, respectively. The putative membrane-spanning domains areunderlined and numbered I to VII. The consensus sequence conservedbetween all the P2Y receptors and the three amino acids (AHN)corresponding to the RGD sequence in the first extracellular loop of theP2Y₂ receptor are represented in bold. The putative phosphorylationsites by PKC or by calmodulin-dependent protein kinases and PKC areindicated respectively by black squares (▪) and by open circles (∘).

FIG. 2 is a dendrogram representing structural relatedness among thecloned P2Y receptor and the closest neighbour in the G protein-coupledreceptor family. The plot was constructed using the multiple sequencealignment program Pileup of the GCG package (26). For each sequence, theanalysis takes into account a segment covering the first five putativemembrane-spanning domains.

FIG. 3 represents a northern blot analysis of P2Y₄ receptor expression.The Northern blot was performed with 15 μg of total RNA from humanplacenta and 4 μg of poly(A)⁺RNA from K562 cells and from two differenthuman placentas. The probe was a human P2Y₄ gene fragment amplified byPCR (TM2 to TM7).

FIG. 4 represents the time course of InsP₃ accumulation in 1321N1 cellsexpressing the human P2Y₄ receptor. ³H inositol labelled cells wereincubated for the indicated time with UTP (100 μM), UDP (100 μM) and ATP(100 μM) in the absence of 10 mM LiCl (panel A) or in its presence(panel B). The data represent the mean of triplicate experimental pointsand are representative of two independent experiments.

FIG. 5 Represents the effect of ATP on the accumulation of InsP₃ inducedby UTP in 1321N1 transfected cells. Concentration-action curves of ATPin the presence of UTP 10 or 100 μM at 30 s (panel A) and 20 min (panelB). Concentration-action curve of ATP with or without UTP (10 μM) at 20min (panel C). The data represent the mean±S.D. of triplicateexperimental points and are representative of two (panel A), five (panelB) or three (panel C) independent experiments.

FIG. 6 represents the concentration-action curves of UTP and UDP on theInSP₃ accumulation in three different clones of 1321N1 transfectedcells. The cells were incubated in the presence of various UTP (●) andUDP (▪) concentrations (0, 0.1, 1, 3, 10 and 100 μM) for 30 s or 20 min.The data represent the mean±S.D. of triplicate experimental pointsobtained in one representative experiment. The EC₅₀ values weredetermined by curve fitting (Sigma Plot: version 2.0).

FIG. 7 Represents the effect of various nucleotides on the InSP₃production in 1321N1 transfected cells.

The cells were incubated with UTP, UDP, 5BrUTP, dUTP, ITP, AP₃A, AP₄A,AP₅A and AP₆A at the same concentration of 100 μM or without agonist(Cont) for 30 s or 20 min. The data represent the mean±S.D. oftriplicate experimental points and are representative of threeindependent experiments. The EC₅₀ values were determined by curvefitting (Sigma Plot: version 2.0).

FIG. 8 Represents concentration-action curves of various nucleotides onthe InsP₃ accumulation in 1321N1 cells expressing a human P2Y₄ receptor.1321N1cells were incubated in the presence of various concentrations ofUTP, UDP, dUTP, 5BrUTP, ITP and ATP for a period of time of 20 min. Thedata are the mean±range of duplicate experimental points obtained in anexperiment representative of two.

FIG. 9 Represents the action of various P₂ antagonists on the InSP₃production induced by UTP in 1321N1 transfected cells. Cells wereincubated in the presence of suramin, reactive blue 2 and PPADS at aconcentration of 100 μM and different UTP concentrations (0, 2 and 10μM) for 20 min. The data represent the mean±S.D. of triplicateexperimental points and are representative of two independentexperiments.

FIG. 10 Represents the effect of PPADS on the UTP stimulation of InsP₃in 1321N1transfected cells. The cells were exposed to variousconcentrations of UTP in the presence or in the absence of PPADS (100μM) for 20 min. The data are the mean±S.D. of triplicate experimentalpoints obtained in an experiment representative of two.

FIG. 11 Represents the effect of pertussis toxin on the UTP-inducedaccumulation of InSP₃ in 1321N1cells expressing a human P2Y₄ receptor.The cells were preincubated for 18 hours in the presence or in theabsence of 20 ng/ml pertussis toxin. The cells were then incubated withor without UTP 100 μM and with or without pertussis toxin (20 ng/ml) forvarious times: 30 s, 5 min or 20 min. The data represent the mean±S.D.of triplicate experimental points and are representative of twoindependent experiments.

DETAILED DESCRIPTION OF THE INVENTION Experimental Procedures

1. Materials

Trypsin was from Flow Laboratories (Bioggio, Switzerland) and theculture media, reagents, G418, fetal calf serum (FCS), restrictionenzymes and Taq polymerase were purchased from GIBCO BRL (Grand Island,N.Y.). The radioactive products myo-D-[2-³H]inositol (17.7 Ci/mmol) and[a³²P]ATP (800 Ci/mmol) were from Amersham (Gent, Belgium). Dowex AG1X8(formate form) was from Bio-Rad Laboratories (Richmond, Calif.). UTP,UDP, ATP, ADP, carbachol, LiCl and apyrase grade VII were obtained fromSigma Chemical Co. (St. Louis, Mo.). 2MeSATP was from ResearchBiochemicals Inc. (Natick, Mass.). pcDNA3 is an expression vectordeveloped by Invitrogen (San Diego, Calif.).

2. Cloning and Sequencing

Degenerate oligonucleotide primers were synthesized on the basis of thebest conserved segments between the murine P2Y2 and the chick P2Y1receptor sequences. These primers were used to amplify novel receptorgene fragments by low-stringency PCR starting from human genomic DNA.The amplification conditions were as follows: 93° C. 1 min, 50° C. 2min, 72° C. 3 min; 35 cycles. The PCR products with sizes compatiblewith P2 receptor gene fragrnents were subcloned in M13mp18 and M13mp19and sequenced by the Sanger dideoxy nucleotide chain termination method.One of the resulting clones sharing similarities with P2 receptors, waslabeled by random priming and used to screen a human genomic DNA libraryconstructed in the λ Charon 4a vector. The hybridization was in 6×SSC(1×SSC: 0.15 M NaCl, 0.015 M Sodium citrate) and 40% formamide at 42° C.for 14 h and the final wash conditions were 0.1×SSC, 0.1% SDS at 65° C.A preparation of λ phages (15) was made for several clones whichhybridized strongly with the probe. A restriction map and a Southernblotting analysis allowed to isolate a 1.4 kb NheI-EcoRV fragment thatwas subcloned into the pBluescript SK⁻ vector (Stratagene). The completesequence of a new receptor coding sequence was obtained on both strandsafter subdloning of overlapping fragments in M13mp18 and M13mp19.

3. Cell Culture and Transfection

The P2Y₄ receptor coding sequence was subdloned between the HindIII andthe EcoRV sites of the pcDNA3 expression vector for transfection into1321N1 human astrocytoma cells, a cell line which does not respond tonucleotides and which has already been used for the expression ofpurinergic receptors (6, 12). Cells were transfected with therecombinant pcDNA3 plasmid (pcDNA3-P2Y₄) using the calcium phosphateprecipitation method as described (16). 1321N1 cells were incubated for6 hours at 37° C. in the presence of pcDNA3 vector alone or vectorcontaining the P2Y₄ receptor coding sequence, then washed and incubatedin culture medium (10% FCS, 100 U/ml penicillin, 100 μ/ml streptomycinand 2.5 μg/ml amphotericin B in Dulbecco's modified Eagle's medium(DMEM)). The selection with G418 (400 μg/ml) was started two days aftertransfection. From the pool of transfected 1321N1 cells, individualclones were isolated by limiting dilution with the aim of selectingclones with high IP stimulation factors in response to nucleotides. Thedifferent clones were maintained in a medium containing 400 μg/ml G418.

4. Inositol Phosphates (IP) Measurement

1321N1 cells were labeled for 24 hours with 10 μCi/ml [³H] inositol ininositol-free DMEM (Dulbecco's modified Eagle's medium) mediumcontaining 5% fetal calf serum, 100 U/ml penicillin, 100 μg/mlstreptomycin, 2.5 μg/ml amphotericin B and 400 μg/ml G418. Cells werewashed twice with KRH (Krebs-Ringer Hepes) buffer of the followingcomposition: (124 mM NaCl, 5 mM KCl, 1.25 mM MgSC₄, 1.45 mM CaCl₂, 25 mMHepes (pH 7.4) and 8 mM glucose) and incubated in this medium for 30 minThe agonists were added in the presence of LiCl (10 mM) and theincubation was stopped after 30 s, 5 min or 20 min by the addition of anice-cold 3% perchloric acid solution. For the time course study, LiCl(10 mM) was added 5 min before the agonists and the incubation wasstopped at different times. When tested, pertussis toxin (20 ng/ml) wasadded for 18 h during the labeling period time and during thestimulation by the agonist. Inositol phosphates were extracted and InsP₃was isolated by chromatography on Dowex column as described previously(17).

5. Radioligand Binding Assay.

Binding assays of [α³²P] UTP to cell membranes were carried out inTris-HCl (50 mM, pH 7.5), EDTA 1 mM in a final volume of 0.5 ml,containing 25-50 μg of protein and 0.5 nM of radioligand (27). Theassays were conducted at 30° C. for 5 min. Incubations were stopped bythe addition of 4 ml of ice-cold Tris-HCl (50 mM, pH 7.5) and rapidfiltration through Whatman GF/B filters under reduced pressure. Thefilters were then washed three times with 2 ml of the same ice-coldTris-HCl buffer. Radioactivity was quantified by liquid scintillationcounting, after an overnight incubation of the filters in liquidscintillation mixture.

6. Northern Blot and Southern Blot Analysis

Total and poly(A)⁺RNA were prepared from different tissues and humancell lines using the guanidinium thiocyanate-cesium chloride procedure(15), denatured by glyoxal and fractionated by electrophoresis on a 1%agarose gel in 10 mM phosphate buffer pH 7.0. DNA samples, prepared fromthe λ Charon 4a clones, were digested with restriction enzymes. Northernand Southern blots were prepared (15) and baked for 90 min at 80° C.Membranes were prehybridized for at least 4 hours and hybridizedovernight with the same probe as for the screening, at 42° C. in asolution containing 50% formamide for Northern blots and 40% formamidefor Southern blots. Filters were washed twice for 15 min in 2×SSC atroom temperature and then twice for 30 min in 0.2×SSC at 60° C. beforebeing exposed at −70° C. in the presence of intensifying screens for 5days (Northern blots) or 1 hour (Southern blots).

Results

1. Cloning and Sequencing

In order to isolate new subtypes of P2 receptors, sets of degenerateoligonucleotide primers were synthesized on the basis of the bestconserved segments in the published sequences of the chick brain P2Y1(5) and murine neuroblastoma P2Y2 (9) receptors. These primers were usedin low-stringency PCR on human genomic DNA as described (18). Somecombinations generated discrete bands with a size compatible with thatexpected for P2 receptors. For example, the primer[5′-CAGATCTAGATA(CT)ATGTT(CT)(AC)A(CT)(CT)T(ACGT) GC-3] (SEQ ID NO:3)corresponding to the second transmembrane region and the primer[5′-TCTTAAGCTTGG(AG)TC(ACG-T)A(CG)(AG)CA(AG)CT(AG) TT-3′] (SEQ ID NO:4)corresponding to the seventh transmembrane region amplified a 712 bpfragment. The partial sequences obtained after sequencing weretranslated into peptidic sequences and compared to a local databankwhich contains G protein-coupled receptor sequences. Most of the clonesresulting from these PCR products encoded a part of a new receptor whichdisplayed 58% identity with the murine P2Y2 receptor and 42% identitywith the chick P2Y1 receptor partial sequences. In addition, some clonesencoded a peptidic sequence presenting 87% identity with the chick P2Y1receptor and are therefore believed to represent fragments of the humanP2Y1 gene.

The partial sequence of the new receptor was used as a probe to screen ahuman genomic DNA library. Several clones that strongly hybridized withthe probe at high stringency conditions were obtained and purified. Theinserts of the clones varied from 12 to 17 kb and restriction analysisrevealed that all clones belonged to a single locus. The full sequenceof a 1.4 kb NheI-EcoRV fragment was obtained and an intronless openreading frame of 1095 bp was identified. The sequence is depicted inFIG. 1 where the putative membrane-spanning domains are underlined andnumbered I to VII. The predicted molecular weight of the encoded proteinis 36.5 kDa. This molecular weight is unlikely to be modified in vivo,since no N-glycosylation consensus sequences are found in the putativeexofacial regions. In contrast with the human P2Y2 receptor, there is noRGD motif, an integrin binding consensus sequence, in the putative firstextracellular loop. The three amino acid (AHN) corresponding to the RGDsequence in the first extracellular loop of the P2Y2 receptor arerepresented in bold in FIG. 1. Some potential sites of phosphorylationby protein kinase C (PKC) or by calmodulin-dependent protein kinaseswere identified in the third intracellular loop and in thecarboxyterminal part of the receptor. The putative phosphorylation sitesby PKC or by calmodulin-dependent protein kinases and PKC are indicatedrespectively by black squares and by open circles in FIG. 1. The fourpositively charged amino acid which have been reported to play a role inthe P2Y2 receptor activation by ATP and UTP (1) are conserved in theP2Y4 sequence: His²⁶², Arg²⁶⁵, Lys²⁸⁹ and Arg²⁹² (FIG. 1). The P2Y4amino acid sequence was compared to the chick P2Y1 and the murine P2Y2amino acid sequences and to their closest neighbours in the Gprotein-coupled receptor family (FIG. 2). The plot was constructed usingthe multiple sequence alignment program Pileup of the GCG package (26).For each sequence, the analysis takes into account a segment coveringthe first five putative membrane-spanning domains. It is clear that,from a structural point of view, the newly cloned receptor is moreclosely related to the human P2Y2 receptor (51% of identity between thecomplete sequences) than to the chick P2Y1 receptor (35%).

2. Tissue Distribution of the P2Y4 Receptor

The tissue distribution of P2Y4 transcripts was investigated by Northernblotting. A number of rat tissues (heart, brain, liver, testis andkidney) were tested using a human probe at low stringency, but nohybridization signal could be obtained. No P2Y4 transcript could bedetected in the following human cell lines: K562 leukemia cells (FIG.3), HL-60 leukemia cells and SH-SY5Y human neuroblastoma cells. TheNorthern blot was performed with 15 μg of total RNA from human placentaand 4 μg of poly(A)⁺RNA from K562 cells and from two different humanplacentas. The probe was the human P2Y4 gene fragment amplified by PCR(TM2 to TM7). On the contrary, a strong signal, corresponding to a 1.8kb mRNA, was found in human placenta (FIG. 3).

3. Functional Expression of the New P2 Receptor in 1321N1 Cells

After transfection of the pcDNA3-P2Y4 construction in 1321N1 cells, thepool of G418-resistant clones was tested for their functional response(IP3 accumulation) to ATP and UTP. Both nucleotides were found to beagonists of the P2Y4 receptor, but the response to UTP was more robust.About 20 transfected clones were then isolated and tested for theirresponse to UTP. The clone presenting the highest IP accumulation factorin response to UTP was selected and used in all subsequent experiments.Functional characterization of the P2Y₄ receptor was performed bydetermining the accumulation of InSP₃ after 20 min incubation with theagonists in the presence of 10 mM LiCl. We observed that the response toUTP was biphasic, with a peak reached at 30 s, followed by a moresustained stimulation of lower magnitude (FIG. 4A). With ATP, only thatsecond phase was detectable: its effect became apparent after 1 min ofstimulation only and was stable for at least 20 min (FIGS. 4A and B). Asfor UTP, the stimulation by UDP was biphasic, but it was slightlydelayed (FIGS. 4A and B). Inclusion of LiCl had little effect on theinitial peak induced by UTP or UDP, but it strongly enhanced thefollowing plateau phase (FIG. 4B).

The maximal effect of ATP observed after a 20 min incubation representedabout 27±9% of that of UTP (mean±S.D. of ten experiments). In order todemonstrate that ATP is able to antagonize the UTP response, incubationsof 1321N1 cells were conducted with ATP alone or in combination withUTP. FIG. 5 shows that at high concentration (500 μM or more), ATP wasable to inhibit the effect of UTP, both at 30 s and 20 min At 30 s, theresponse to UTP 10 μM was fully antagonized by ATP 2 mM, correspondingto the fact that ATP has no effect on the human P2Y₄ receptor at thisearly time (panel A). At 20 min, an inhibition of 62±11% of the UTPeffect (10 μM), corresponding to the difference between the UTP and theATP effects, was observed in the presence of 2 mM ATP (mean±S.D. of fiveindependent experiments) (panels B and C). The ATPconcentration-inhibition curves were shifted to the right when the UTPconcentration was increased, indicating the competitive nature of thisinhibitory effect (panels A and B). On the other hand, at lowerconcentrations (30-300 μM), ATP enhanced the response to UTP by 29%(range 12-47%, mean of four experiments) (panel B). ADP, which hadalmost no effect per se and did not inhibit the action of UTP,reproduced that enhancement: in the presence of ADP (100 μM), thestimulation by UTP (10 μM) represented 158±15% (mean of threeindependent experiments) of that by UTP alone (data not shown). However,this potentiating effect of ATP and ADP was not specific: indeed theaction of carbachol mediated by muscarinic receptors endogenouslyexpressed in the 1321N1 cells (6) was also increased in the presence ofthese nucleotides. This observation was reproduced with cellstransfected with the recombinant P2Y₄-pcDNA3 plasmid or with the vectoralone and was also obtained with AMP and adenosine (data not shown).

We compared the concentration-action curves of UTP and UDP on the InSP₃production for several clones of transfected cells. The study was madeat two times (FIG. 6): 30 s and 20 min. In the set of experimentsperformed on clone 11 (clone of 1321N1 transfected cells chosen for thepharmacological characterization), UTP appeared to be 10-fold morepotent than UDP after a 20 min incubation and this difference wasreproduced with two other clones (FIG. 6). The EC₅₀ values were 0.3±0.1μM and 3.3±0.6 μM in clone 2, 2.4±0.1 μM and 19.8±4.8 μM in clone 11 and0.3±0.1 μM and 3.2±0.8 μM in clone 21, respectively, for UTP and UDP(mean±S.D. of two independent experiments). At 30 s of incubation, itwas not possible to determine EC₅₀ values because the curves wereclearly shifted to the right, but we can observe that the differencebetween the two agonists potency was even more striking (FIG. 6).Several clones, including clones 2, 11 and 21 were tested in bindingstudies with [α³²P] UTP but no increase in specific binding was observedas compared to the cells transfected with the vector alone (data notshown).

In view of the time differences observed in FIG. 6, the testing of arange of nucleotides was performed at two times: 30 s and 20 min. AsFIG. 7 shows, several agonists were barely or not active at 30 s (UDP,5BrUTP, dUTP, ITP) whereas they produced a significant effect at 20 min.Full concentration-action curves were obtained at 20 min. The rank orderof potency was: UTP>UDP=dUTP>5BrUTP>ITP>ATP (FIG. 8). The EC₅₀ valuesobtained were the following: EC₅₀UTP=2.5±0.6 μM, EC₅₀ UDP=19.5±3.9 μM(mean±S.D. of eight independent experiments), EC₅₀ dUTP=20.0±2.3 μM,EC₅₀ 5BrUTP=27.1±1.9 μM and EC₅₀ ITP=32.8±5.4 μM (mean±S.D. of twoindependent experiments). The approximative EC₅₀ value obtained for ATPwas: 43±12 μM (mean±S.D. of five independent experiments). Thediadenosine polyphosphates also increased the InSP₃ production intransfected cells with EC₅₀ between 3 and 7 μM (data not shown), buttheir maximal effect was only 20-25% of that of UTP, a value close tothat of ATP (range of four independent experiments) (FIG. 7). UMP,uridine, AMP, adenosine and ATPyS were without any effect (data notshown).

No specific antagonist is available for any P2Y subtype. Nonetheless,several non-selective antagonists such as suramin, RB2 or PPADS havebeen tested on P₂ receptors and their relative actions on these subtypesmay constitute a mean to discriminate them (27). So we tested theability of these three antagonists to inhibit the UTP response in themodel of the human P2Y₄ receptor. As we can see on FIG. 9, PPADSappeared to be the most active antagonist (73±14% inhibition; IC₅₀around 15 μM (data not shown)), suramin was inactive, and RB-2 producedan inhibition of 33±5% of the UTP response (mean±S.D. of two independentexperiments). FIG. 10 shows the mixed nature of the antagonism by PPADSof the UTP response: it affects both the EC₅₀ value and the maximaleffect of UTP. The EC₅₀ value for UTP in the absence of PPADS was3.3±0.6 μM and 12.2±4.5 μM in the presence of 100 μM PPADS (mean±S.D. oftwo independent experiments).

The effect of pertussis toxin (20 ng/ml, 18 hours pretreatment) wasstudied at different times after UTP (100 μM) addition (FIG. 11). TheUTP response was clearly inhibited at 30 s (62±5% of inhibition:mean±S.D. of two independent experiments), whereas no significant effectwas observed at 5 and 20 min.

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All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1. A method for determining whether a ligand can specifically bind to areceptor, wherein said receptor comprises the amino acid sequence shownin SEQ ID NO:2, comprising the following steps: i) preparing a cellextract from cells transfected with an expression vector comprising thenucleic acid molecule encoding said receptor, ii) isolating a membranefraction from the cell extract, iii) contacting the ligand with themembrane fraction under conditions permitting binding of the ligand tosaid receptor, and iv) detecting the presence or absence of the ligandbound specifically to said receptor, wherein the presence of the ligandindicates that the ligand can specifically bind to said receptor.
 2. Themethod of claim 1, wherein said receptor consists of the amino acidsequence shown in SEQ ID NO:2.